Tissue transglutaminase promotes drug resistance and invasion by inducing mesenchymal transition in mammary epithelial cells

Abstract

Recent observations that aberrant expression of tissue transglutaminase (TG2) promotes growth, survival, and metastasis of multiple tumor types is of great significance and could yield novel therapeutic targets for improved patient outcomes. To accomplish this, a clear understanding of how TG2 contributes to these phenotypes is essential. Using mammary epithelial cell lines (MCF10A, MCF12A, MCF7 and MCF7/RT) as a model system, we determined the impact of TG2 expression on cell growth, cell survival, invasion, and differentiation. Our results show that TG2 expression promotes drug resistance and invasive functions by inducing epithelial-mesenchymal transition (EMT). Thus, TG2 expression supported anchorage-independent growth of mammary epithelial cells in soft-agar, disrupted the apical-basal polarity, and resulted in disorganized acini structures when grown in 3D-culture. At molecular level, TG2 expression resulted in loss of E-cadherin and increased the expression of various transcriptional repressors (Snail1, Zeb1, Zeb2 and Twist1). Tumor growth factor-beta (TGF-β) failed to induce EMT in cells lacking TG2 expression, suggesting that TG2 is a downstream effector of TGF-β-induced EMT. Moreover, TG2 expression induced stem cell-like phenotype in mammary epithelial cells as revealed by enrichment of CD44(+)/CD24(-/low) cell populations. Overall, our studies show that aberrant expression of TG2 is sufficient for inducing EMT in epithelial cells and establish a strong link between TG2 expression and progression of metastatic breast disease.

Figure 1. TG2 induces EMT in mammary epithelial cells.

MCF10A and MCF12A cells were stably transfected with vector alone (10A-Vec and 12A-Vec) or lentiviral-TG2 construct (10A-TG2 and 12A-TG2). (A) Phase-contrast images of 10A-Vec and 10A-TG2 cells after 48 hr culture in medium. Magnification 10X. (B) Immunoblot showing relative expression of TG2 in 10A-Vec and 10A-TG2 cells. (C) Immunofluorescence due to TG2 and EMT markers in 10A-Vec (top panel) and 10A-TG2 (bottom panel) cells. Green fluorescence shows the expression and localization of indicated proteins and DAPI (blue) staining shows the nuclei. (D) Immunoblot analysis of the indicated EMT markers in 10A-Vec and 10A-TG2 cells. Expression of epithelial cell markers (E-cadherin, β-catenin) and mesenchymal cell marker (N-cadherin, fibronectin, and vimentin) was examined by immunoblotting. (E) Morphology (left penal) and immunoblot analysis of TG2, epithelial marker (E-cadherin) and mesenchymal marker (fibronectin) expression in 12A-vec and 12A-TG2 cells. (F) Morphology (left penal) and immunoblot analysis for constitutive TG2, epithelial marker (E-cadherin) and mesenchymal marker (fibronectin) expression in drug-sensitive and drug-resistant (RT) MCF-7 cells. Multiple stable polyclones were established from MCF10A and MCF12A cells and experiments were repeated multiple times with similar results using different clones.

Figure 2. TG2 expression results in altered expression of Snail1, Twist1, Zeb1, and Zeb2.

(A) Real time RT-PCR array showing relative changes in the expression of EMT-related genes in 10A-TG2 cells in comparison to 10A-Vec cells. Y-axis denotes the fold- expression and x-axis denotes the genes. The expression of GAPDH, β-actin and 18S ribosomal RNA was used to normalize variable template loading. (B) RT-PCR analysis for EMT-related transcripts was used to validate their expression in 10A-Vec and 10A-TG2 cells. (C) RT-PCR (left panel) and immunoblot (right panel) analysis was performed to validate the expression of transcription factors Snail1, Zeb1 and Twist1 in 10A-Vec and 10A-TG2 cells. Results shown are from a representative experiment repeated at least twice with similar results.

Figure 3. TG2-induced EMT confers invasiveness, drug resistance, and tumorigenic phenotype.

(A) Transwell-Matrigel invasion assay was performed with 10A-Vec and 10A-TG2 cells. Cells that invaded though the Matrigel after 72 hr incubation were counted in five random microscopic fields under 20X magnification. Experiments were done three times in triplicate. Results shown are the average number of invading cells per field ± SEM. (B) Graph represents average number of colonies formed from three independent experiments ± SEM after 3 weeks' incubation of cells in soft agar. Images of colonies formed from a representative experiment after 3 weeks culture in soft agar are shown in right panel. Magnification 10X. (C) Phase-contrast images of acinar structures (4 and 12 days) formed as a result of 10A-Vec and 10A-TG2 cell culture in Matrigel-coated glass-slide chambers for indicated time periods. Inset, amplified view of individual MCF10A-TG2 cells invading the surrounding Matrigel (indicated by the arrow). (D) Loss of basement membrane integrity and cell-to-cell adhesion in 10A-TG2 acini. MCF10A cells were cultured in Matrigel-coated chambers for 12 days and immunostained for laminin V and E-cadherin (green) and DAPI (blue). Representative images from two independent experiments with similar results are shown. Magnifications 20X. (E) Sensitivity of 10A-Vec and 10A-TG2 cells to doxorubicin. Quadruplicate wells in 96-well plates, containing 2,000 cells per well in 0.2 ml of the complete medium (10% FCS) were either left untreated or treated with indicated concentrations of doxorubicin. Two days after the treatment, viable cells remaining in wells were determined by MTS reduction test, and percent cell viability was calculated. Experiments were repeated at least three times with similar results. Bars, mean of quadruplicate values from a representative experiment; lines, SD.

Figure 4. TG2 is a downstream mediator of TGF-β-induced EMT.

(A) Supershift assay for NF-κB activity using EMSA with the nuclear extracts prepared from indicated cells. Nuclear extracts were incubated with an anti-p65 antibody and anti-p50 antibody, or nonradioactive (cold) or mutant NF-κB oligonucleotides and examined for DNA binding. Nuclear extract of KMB cells treated with TNF-α (+TNF) were used in parallel as positive control. (B) Immunoblot analysis showing the level for pAKT(S473) and pFAK(Y397) in 10A-Vec and 10A-TG2 cells. (C) Phase-contrast images of MCF10A cells transfected with control-shRNA or TG2-shRNA and incubated with 2.5 ng/ml recombinant TGF-β for indicated time periods. After 8 and 12 days of TGF-β-treatment MCF10A-control-shRNA cells showed mesenchymal morphology but not the TG2-shRNA transfected cells. (D) Immunoblot analysis for expression of TG2, E-cadherin and fibronectin in MCF10A-control-shRNA and MCF10A-TG2-shRNA cells in response to TGF-β treatment at indicated time points. (E) Downregulation of endogenous (MCF-7/RT) or induced (MCF10A-TG2) TG2 by siRNA resulted in loss of fibronectin (mesenchemyal marker) and upregulation of E-cadherin (epithelial marker) expression. Results shown are from a representative experiment repeated twice with similar results.

Figure 5. TG2-induced EMT promotes stem cell-like phenotype.

FACS analysis of cell surface markers CD326, CD45, CD44 and CD24 in MCF10A (A) and MCF-7 (B) breast epithelial cells, expressing exogenous (10A-TG2) or endogenous (MCF-7/RT) TG2. 10A-Vec and MCF-7 cells, which lack TG2 expression, served as controls. Results shown are from a representative experiment repeated multiple times on different occasions.

Figure 6. Schematic of TG2-indued pathways involved in promoting the metastatic phenotype.

Inflammatory signals such as generation of ROS^-, hypoxia, or TGF-β induce the expression of TG2 in epithelial cells. Induction of TG2 results in constitutive activation of AKT, FAK and NF-κB, which can lead to the transcriptional regulation of Snail1, Zeb1, and Twist. Expression of these transcription factors represses the E-cadherin and induces the expression of fibronectin, N-cadherin and vimentin. These changes result in transformation of immotile epithelial cells to motile mesenchymal cells resulting in altered cell-cell (homotypic) and cell-ECM (heterotypic) interactions and offers metastatic niche to the cells in terms of increased invasiveness, survival and self-renewing capacity by conferring stem cell-like phenotype. TG2 expression also promotes anchorage-independent growth.